By measuring the fluorescence emission spectra of individual leaf sections (with or without leaf-veins) in a
conventional fluorometer it could be demonstrated that the blue-green fluorescence emission, which is known to
originate primarily if not exclusively from the cell walls (Stober and Lichtenthaler, 1993a), mainly emanates from the
main leaf-vein and to a lower degree also from the lateral leaf-veins. The differences in the fluorescence yield
between intercostal fields and leaf-veins are found in leaves of green and of aurea tobacco. That the intensity of the
the emitted blue-green fluorescence (F440, F520) and the red chlorophyll fluorescence F690 and F735 are different
between green and aurea tobacco is due to their differential content of chlorophylls and carotenoids and to a different
degree of reabsorption of the fluorescences by the absorption bands of chlorophyll-carotenoid-proteins in the
photochemically active thylakoids of chloroplasts. Since these photosynthetic pigments and pigment-proteins are
unevenly distributed within the bifacial leaves of the C3*plant tobacco, the uneven distribution of the blue-green and
red fluorescence signatures and ratios (Table 1) between the two leaf-sides (upper and lower) as well as between leafveins
leafveins and intercostal fields is to be expected and fully understandable.
The data also demonstrate that the fluorescence emission spectra measured with a conventional fluorometer of a
particular leaf region is a combined signal which is composed of the relative blue-green and red fluorescence
characteristics of the intercostal fields and leaf-veins. Depending upon the relative amounts of lateral or main leafveins
leafveins of the measured leaf surface, the blue-green fluorescence is high or low with respect to the red chlorophyll
fluorescence (Fig. 2A). The former conclusion, that the cells of the leaf-veins with their predominantly chlorophylland
chlorophylland carotenoid-free cells would contribute much more to the blue-green fluorescence emission of leaves than the
vein-free leaf parts (Stober and Lichtenthaler, 1993 a), was thus experimentally proved in this investigation for the
first time.
At a first view there appears to be general agreement of the results of fluorescence images of the tobacco leaves for
the four fluorescence emission regions blue, green, red and far-red with the results obtained for leaf pieces using the
conventional fluorometer. In both cases we could prove the much higher blue-green fluorescence emission of leafveins
leafveins than in the intercostal leaf fields. Since the fluorescence images exhibit a much better resolution and allow to
differenciate clearer between the main or lateral leaf-veins and the surrounding green leaf tissue, they are preciser
concerning the origin of blue-green and red fluorescences over the leaf surface. In contrast to the conventional
spectrofluorometer, where the main leaf-vein not only showed a high blue fluorescence but also a considerable red
chlorophyll fluorescence (Figs. 1 and 2) from the neighbouring green leaf parts, the fluorescence images
demonstrated that the leaf-veins exhibit a high blue fluorescence, but a very low red chlorophyll fluorescence
emission (Fig. 3). That blue-green fluorescence and red chlorophyll fluorescence emission are in a negative or inverse
contrast to each other, has been demonstrated here for the first time.
Fluorescence images have the advantages to allow the determination of the overall fluorescence distribution over the
surface of a whole leaf very precisely in one single measurement. Fluorescence imaging spectroscopy also permits to
present leaf images via the different fluorescence ratios for each point of a leaf (Fig. 4) as well as the average ratio for
the whole leaf. These are interesting possibilities with respect to the remote sensing of the difference of the pigment
content of plants and detecting stress to leaves and whole plants. Laser-induced fluorescence imaging spectroscopy
thus may prove to be a superior technique for the future remote sensing of plants and terrestrial vegetation.
ACKNOWLEDGEMENTS
Part of this work was sponsored by a grant from BMFT (Germany) and MRE (France) within the EUREKA
LASFLEUR Project (EU 380) which is gratefully acknowledged.
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